Post-translational modifications of proteins are often counteracted by reactions that erase them, and conjugation of proteins with the small protein modifier ubiquitin (Ub) is no exception. The role of Ub as a post-translational modifier includes the control proteasomal proteolysis, involvement in receptor and membrane trafficking, as well as participation in the regulation of gene expression. A complex cascade of diverse enzymatic activities controls the addition of Ub. The outcome of these reactions is offset by deubiquitinating enzymes (DUBs), an enzyme family that rivals in diversity that of the Ub ligases. Defects in these pathways are associated with a diverse set of diseases. The possible exploitation of DUBs as targets for pharmacological intervention requires a detailed understanding of the functional properties and specificity of this enzyme class. To dissect the functional role of these proteases, we propose to develop new probes, based on installation of electrophilic substituents on the C-terminus of Ub to capture and identify active DUBs. These probes will be used to profile the expression/activity of DUBs during microbial infections and the changes therein upon lymphocyte activation. We further aim to identify new small-molecule, cell permeable inhibitors in an approach that includes high throughput screens. With activity-based probes and small molecule inhibitors of DUBs in hand, we shall design peptide libraries in isopeptide linkage to the C- terminus of Ub to define the specificity of DUBs. We shall further endeavor to identify proteins that interact with DUBs as entities that serve as substrates or that perform regulatory functions and control enzyme activity. Combined, these approaches will allow the positioning, by chemical and functional analysis, of a complex family of DUBs in their proper physiological context.